Mutagenesis of linear or circular DNA larger than five to eight kbp (kilobase pair) in size is a tedious and often slow process. Most mutagenesis technologies for such DNA require the availability of unique restriction enzyme sites flanking the region targeted for mutagenesis and numerous steps of DNA and bacterial handling. Some of these steps include excising a fragment of DNA that contains the mutagenesis target site along with a flanking region, hereafter referred to as the target-containing DNA fragment (TDF), using restriction enzyme(s), followed by inserting the TDF into a cloning plasmid which is usually less than five kbp in size. This step requires cleaving both the original plasmid containing the TDF and the cloning plasmid with restriction enzymes and subsequently ligating the TDF into the cloning plasmid.
The TDF-containing cloning plasmid is then transformed into bacterial cells, and the transformed bacteria are grown on selective solid medium to allow only those bacterial cells that successfully uptake the TDF-containing cloning DNA plasmid to grow into colonies. Not all colonies will contain the newly introduced TDF because some of the empty cloning plasmids can transform bacteria due to inefficiency in restriction enzyme cleavage. Therefore, several bacterial clones are picked from the solid medium plates and grown in liquid medium. Their DNA contents are extracted with chemical reagents and multiple purification steps are required to remove contaminating bacterial chromosomal DNA, RNA, proteins and carbohydrates. The extracted DNA is then analyzed by DNA sizing, restriction analysis or PCR methods to identify a correct TDF-containing clone. Once a correct bacterial clone is identified, it is amplified by growing it in a larger volume of bacterial growth liquid medium, harvested by centrifugation, lysed by chemical reagents, and its plasmid DNA content is extracted and purified. The actual mutagenesis steps are then performed on this plasmid DNA by conventional methods such as site-specific mutagenesis without phenotypic selection (Kunkel, Proc Natl Acad Sci USA 82(2):488-92, 1985), inverted PCR (Byrappa et al., Genome Res 5(4), 404-7, 1995), the QuikChange XL site-directed mutagenesis kit (Stratagene, U.S. Pat. Nos. 5,789,166 and 5,932,419), or similar methods.
After completing the mutagenesis, a mutant clone must be identified by repeating the bacterial transformation, growth on solid medium, selection of colonies, growth in medium, and DNA purification. At this stage and depending on the nature of the introduced mutation, a DNA sequencing step is usually necessary to ensure that the selected clone contains the correct mutation. The correct clone is then grown in liquid bacterial growth medium followed by centrifugation to harvest the bacterial pellet, and the plasmid DNA is extracted. The authenticity of the resulting DNA is examined by restriction analysis using the same restriction enzyme(s) that was used to excise the TDF from the Original DNA and insert it into the cloning plasmid, or other restriction enzymes that result in a unique pattern. The mutated TDF is then cleaved out of a correct plasmid by restriction enzymes and separated from the cloning plasmid backbone. This separation step is usually performed on agarose or polyacrylamide gels in the presence of a fluorescent dye to visualize and physically collect the mutated TDF. Gel-purified, mutated TDF is then ligated to the Original plasmid that had been cleaved with the same restriction enzymes used to isolate the TDF and purified on agarose gel separately. Each of the steps described above is time consuming, laborious, and can be fraught with problems that slow the mutagenesis process.
There is a need to develop more efficient, economical and reliable methods for modifying large DNA accurately and with a minimum number of steps. The invention described below, Unrestricted Mutagenesis and Cloning (URMAC), avoids all of the steps described above for conventional mutagenesis methods until insertion of the mutated TDF into the Original DNA. Instead, URMAC depends on a series of PCR reactions, punctuated by ligation reactions. URMAC allows precise modification of linear or circular DNA of any size at any point. In its simplest form, URMAC uses two unique restriction sites flanking the TDF in the Original DNA that are in close enough proximity to each other so that a fragment containing these restriction sites can be amplified by PCR. In a variation of URMAC that employs homologous recombination, flanking restriction sites are not necessary.